Screening And Function Of A Two-component System For Drug Resistance Of Salmonella Typhimurium CVCC541

Salmonella Typhimurium,a Gram-negative intestinal bacillus,is the pathogen of the disease of salmonellosis which has a huge impact on human health,animal husbandry and aquaculture.In recent years,the abuse of antibiotics lead to the phenomenon of multidrug resistance,and the prevention and treatment of salmonellosis become more difficult.Bacteria need to regulate their own metabolism to adapt to various stimuli in order to survive in the environment.Among them,the most important regulatory system is the two-component system?TCS?of the bacteria,which is involved to a variety of bacterial biological functions and is a signal transduction pathway for phosphate transfer.It is reported that there are more than 20 types of Salmonella two-component system.In this study,the TCS including Arc A/ArcB,Kdb D/KdpE,RssA/RssB,BaeS/BaeR,PmrA/PmrB,PhoP/PhoQ,RstA/RstB,UvrY,NtrB,TtrS/TtrR,QseB/Qse C,NarL/NarX,Dcu S/DcuR,CreC/CreB,GlrR/GlrK,BarA/SirA,and TorR systems of Salmonella typhimurium CVCC541,was screened for their drug-resistance by the construction of the above-mentioned system gene deletion strains.Red homologous recombination technology was used to construct two-component gene deletion strain of Salmonella typhimurium CVCC541,and the minimum inhibitory concentration?MIC?of the mutant strains to antibiotics was determined.PCR and sequencing was used to identify the correct construction of the two-component system gene deletion strains of Salmonella typhimurium CVCC541.The MIC test showed that compared with the parent strain,deletion of pmrA and pmrB result in 8-fold and 16-fold increase in sensitivity to polymyxin B,respectively;deletion of phoP and phoQ result in8-fold increase in sensitivity to polymyxin B and 4-fold increase in sensitivity to doxycycline,deletion fo phoQ result in 4-fold increase in sensitivity to penicillin G;deletion of arcA and arcB result in16-fold and 8-fold increase in resistance to streptomycin,respectively,and deletion of arcA result in8-fold increase in resistance to neomycin,deletion of arcB result in 8-fold increase in resistance to amikacin;deletion of creC result in 8-fold increase in resistance to tetracycline.It is confirmed that the two-component system regulating drug resistance include PmrAB,PhoPQ,Arc AB and CreCB systems.Among them,PmrAB and PhoPQ systems regulate the resistance to polymyxin and ArcAB system regulates the resistance to aminoglycosides,the CreCB system regulates resistance to tetracyclines.The regulatory mechanism of ArcAB system involved drug resistance is not yet clear to date.The analysis of the structure of the arcA and arcB genes in this system showed that the Asp⁵66 locus of the arcA gene and the His²⁹²,Asp⁵⁷⁶,and His⁷1717 loci of the arcB gene are the key sites of its phosphorylation signal transduction.Therefore,in this study,the pSTV28 plasmid was used to construct CVCC541?arcA/parcA,CVCC541?arcB/parcB,and the point mutation mutants arcA^Asp-56-Ala,arcB^His-292-Ala,arcB^Asp-576-Alasp-576-Ala and arcB^His-717-Alais-717-Ala were constructed by seamless cloning and recombinant technology.PCR and sequencing indicated that the correct construction of replenishing strains and point mutant replenishing strains.The biological characteristics tests showed that the growth of arcA,arcB mutant and point mutation replenishing strains was slower than the parent strain in vitro,and the growth of arcA replenished strain and arcB replenished strain was consistent with the parent strain,indicating that the deletion of the gene has a certain effect on the growth of CVCC541.The MIC tests showed that CVCC541?arcA and CVCC541?arcB had different degrees of sensitivity to aminoglycoside drugs;All replenished strains showed similar drug susceptibility compared to the parental strain.The key site mutation of arcA and arcB gene is consistent with the regulation of arcA and arcB knockout on the aminoglycoside resistance of Salmonella typhimurium CVCC541.The study indicated that the arcA and arcB genes was involved in the regulation of aminoglycoside antibiotic resistance of Salmonella typhimurium CVCC541 via phosphorylation pathway.